: Spin-mini. Spin-mini,, (13-Desmethyl Spirolide C, SPX1) 3.0~40.0 μgkg -1, 14.3., (LC-MS/MS) , LC-MS/MS. (MRL),, (AZAs) 160.
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- Ὡρος Αλεξιάδης
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1 313, Vol.31 No.3, May 2018 JOURNAL OF NINGBO UNIVERSITY ( NSEE ) 1, 1, 2, 2, 1*, 1, 1 1., ; 2., : Spin-mini. V():V():V()=7:2:1, Spin-mini,,. -2(PTX2)(Gymnodimine, GYM) 3.0~ 30.0 μgkg -1, (Azaspiracids, AZA1AZA2AZA3) 0.75~10.0 μgkg -1, (13-Desmethyl Spirolide C, SPX1) 3.0~40.0 μgkg -1, , 0.10~0.26 μgkg -1, 0.28~0.81 μgkg ~115.7, 0.9~ 14.3.,. : ; ; ; : O : A : , (), [1],,,, [2-4].,,,,, [5-9].,, (MRL),, (AZAs) μgkg -1, -2 (PTX2) μgkg -1, (SPXs) [10-11]., [12-15]. 2011,, (LC-MS/MS),,.,,, LC-MS/MS [16-19]. (Solid Phase Extraction, SPE), [20-22]. SPE, : ; ; ; 2015C110018; XKZSC1515, XKZSC ,,,, :. chaijiye@163.com * 1979,,, /, :. shixizhi@nbu.edu.cn
2 ,.,, (Spin-mini),,,, LC-MS/MS (PTX2GYMAZA1AZA2AZA3 SPX1) National Research Council, Halifax, NS, Canada; : HPLC, >99.9, Thermo Fisher Scientific, USA; HPLC, >99.9, Tedia Company, USA; : HPLC, >99.5, Honeywell, USA; :, >99.7, ;, 25~28, ; : ELGA, 18.2 MΩ; Nanoinnova Technologies Company, Spain. Waters (ACQUITY UPLC) (XEVO-TQ), (ESI)(APCI) (Waters, USA); : Waters XBridge C 18 Column (5.0 µm, mm 2.1 mm i.d.); (PT2000, Switzerland); (Type 3K-18, Sigma, Germany); (SCILOGXE MX-S, Scilogex, USA); (IKA T-18, Ika, Germany); (N-EVAP-24, Oragnic Association, USA); (SK250H, ) PTX2GYMSPX1AZA1 AZA2AZA3,, PTX2GYMSPX μgl -1 ; AZA1AZA2AZA μgl -1, ,,, rmin -1 2 min, 80 C. (2.00±0.01) g 15.0 ml, 2.0 ml V():V(): V()=7:2:1, 30 s, g 5min, 15.0 ml. 1., 20 C, 2h,, 40 C, 1.0 ml 30, Spin-mini Spin-mini,, 30.0 mg,,, Spin-mini ( 1) Spin-mini 1 ; 2 ; 3 ; 4. 1 Spin-mini 1.0 ml, Spin-mini, 30.0, 1.0 ml, 1.0 ml 30.0, g 10 min, 0.5 ml V(): V():V()=7:2:1, rmin min,, UPLC-MS/MS Waters XBridge C 18 (5.0 µm, mm 2.1 mm i.d.); 0.15 ( A)( B). : 40 C; : 10.0 μl; : A 0.15, B ; 0.3 mlmin -1. :, 1. : (ESI), ; : (MRM); : 145 ; : 450 ; : 750 Lh -1 ; : 50 Lh -1 ; : V; 4
3 3, : 67 : ESI kvesi 3.0 kv /(mlmin -1 ) A/ B/ (m/z) (m/z) /V /ev GYM [M-H] * AZA1 [M-H] * AZA2 [M-H] * AZA3 [M-H] * SPX1 [M-H] * PTX2 [M-H] * : * , V():V():V()=7:2:1V():V( ):V( )=2:7:1 V( ):V( ):V( )=4.5:4.5:1 6, 2. 2, V():V( ):V()=7:2:1, ~102.6, 6., V():V():V()=7:2: GYM PTX2 AZA1 AZA2 AZA3 SPX1 1 ; 2 ; 3 ; 4 V():V():V( )=7:2:1; 5 V():V():V()=2:7:1; 6 V():V():V()=4.5:4.5: Spin-mini Spin-mini,., 10.0~50.0/, 3. 3, 10.0~30.0, ; 40.0, PTX2, 50.0, 6.,, ,,.,, GYM PTX2 AZA1 AZA2 AZA3 SPX , 1.0 ml ,, , 30.0.,, 30.0.,
4 , () (V():V():V( )=7:2:1), μl, 1. 3, 3. 3, (V( ):V( ):V( )=7:2:1)(92.8~102.9) (<10.9). 2.3.,. XBridge C 18 (Waters, 5.0 µm, mm 2.1 mm i.d.) 0.15( A)( B),., (MS/MS),, 2.,, 6 ( 4), XBridge C 18, Spin-mini SPE UPLC-MS/MS., / (μgkg -1 ) 3 3(RSD) 0.15 V():V():V()=7:2:1 / RSD/ / RSD/ / RSD/ PTX GYM AZA AZA AZA SPX (a) PTX μgkg -1 (b) GYM 10.0 μgkg -1 (c) SPX μgkg -1 (d) AZA1 2.5 μgkg -1 (e) AZA2 2.5 μgkg -1 (f) AZA3 2.5 μgkg
5 3, : 69 4 /(μgkg -1 ) PTX2 3.00~ x x GYM 3.00~ x x AZA1 0.75~ x x AZA2 0.75~ x x AZA3 0.75~ x x SPX1 3.00~ x x ,, ESI,, [23-26]..,,,, 4. AZA1AZA2AZA3SPX1 0.8~1.2,, PTX2 GYM ,., 4, 6, ,, , (S/N) 10:1,, 0.28~0.81 μgkg -1 ; S/N 3:1,, 0.10~0.26 μgkg -1, , 6. Spin-mini UPLC-MS/MS, ~115.7, 14.3,. 5 6 μgkg -1 PTX2 3.00~ GYM 3.00~ AZA1 0.75~ AZA2 0.75~ AZA3 0.75~ SPX1 3.00~ (n=3) (n=9) /(μgkg -1 ) / RSD/ / RSD/ PTX GYM AZA AZA AZA SPX (Mytilus edulis)
6 (Ostrea gigas thunberg)(meretrix meretrix L) (Scapharca subcrenata)(ruditapes phillppinarum)5,., 2 GYM, 0.3 μgkg μgkg -1 ; 2 SPX μgkg μgkg -1,,. 3 Spin-mini UPLC-MS/MS 6., 30.0, V():V():V()=7:2:1, ~115.7, 0.9~14.3, 0.10~0.26 μgkg ~0.81 μgkg -1.,. [1],,,. [J]., 2014, 33(3): [2],,,. [J]., 2016, 35(5): [3],,,. [J]., 2014, 36(3): [4],,,. [J]., 2013, 20(2): [5],,,. [J]., 2016, 28(1): [6],,,. [J]., 2014, 45(4): [7] Li X, Li Z, Chen J, et al. Detection, occurrence and monthly variations of typical lipophilic marine toxins associated with diarrhetic shellfish poisoning in the coastal seawater of Qingdao City, China[J]. Chemosphere, 2014, 111: [8] Chen J, Li X, Wang S, et al. Screening of lipophilic marine toxins in marine aquaculture environment using liquid chromatography-mass spectrometry[j]. Chemosphere, 2017, 168: [9] Bosch-Orea C, Sanchis J, Farre M, et al. Analysis of lipophilic marine biotoxins by liquid chromatography coupled with high-resolution mass spectrometry in seawater from the Catalan Coast[J]. Analytical and Bioanalytical Chemistry, 2017, 409:1-12. [10] Hess P. Requirements for screening and confirmatory methods for the detection and quantification of marine biotoxins in end-product and official control[j]. Analytical and Bioanalytical Chemistry, 2010, 397(5): [11] García-Mendoza E, Sánchez-Bravo Y A, Turner A, et al. Lipophilic toxins in cultivated mussels (Mytilus galloprovincialis) from Baja California, Mexico[J]. Toxicon, 2014, 90: [12] Rodríguez L P, González V, Martínez A, et al. Occurrence of lipophilic marine toxins in shellfish from Galicia (NW of Spain) and synergies among them[j]. Marine Drugs, 2015, 13(4): [13] Hess P, Butter T, Petersen A, et al. Performance of the EU-harmonised mouse bioassay for lipophilic toxins for the detection of azaspiracids in naturally contaminated mussel (Mytilus edulis) hepatopancreas tissue homogenates characterised by liquid chromate-graphy coupled to tandem mass spectrometry[j]. Toxicon, 2009, 53(7/8): [14] Liu B H, Hung C T, Lu C C, et al. Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographicstrip[j]. Journal of Agricultural and Food Chemistry, 2014, 62(6): [15] Lian Z R, Wang J T. Study of molecularly imprinted solid-phase extraction of gonyautoxins 2, 3 in the cultured dinoflagellate Alexandriumtamarense by highperformance liquid chromatography with fluorescence detection[j]. Environmental Pollution, 2013, 182: [16] Mattarozzi M, Milioli M, Bianchi F, et al. Optimization of a rapid QuEChERS sample treatment method for HILIC-MS 2 analysis of paralytic shellfish poisoning (PSP) toxins in mussels[j]. Food Control, 2016, 60: [17] Kilcoyne J, Mccarron P, Twiner M J, et al. Epimers of azaspiracids: Isolation, structural elucidation, relative LC- MS response, and in vitro toxicity of 37-epi-azaspiracid-1[J].
7 3, : 71 Chemical Research in Toxicology, 2014, 27(4): [18],,,. [J]., 2016, 34(4): [19], 珮,,. QuEChERS 3 [J]., 2013, 31(10): [20] Raterink R J, Lindenburg P W, Vreeken R J, et al. Recent developments in sample-pretreatment techniques for mass spectrometry-based metabolomics[j]. TrAC Trends in Analytical Chemistry, 2014, 61: [21],,,. [J]., 2016, 37(6): [22],,,. [J]., 2014, 35(8): [23] Sjin C, Jang H, Jo H, et al. Development and validation of an accurate and sensitive LC-ESI-MS/MS method for the simultaneous determination of paralytic shellfish poisoning toxins in shellfish and tunicate[j]. Food Control, 2017, 77: [24] Mccarron P, Reeves K L, Giddings S D, et al. Development of certified reference materials for diarrhetic shellfish poisoning toxins, Part 2: Shellfish matrix materials[j]. Journal of AOAC International, 2016, 99(5): [25] Shen Q, Gong L, Baibado J T, et al. Graphene based pipette tip solid phase extraction of marine toxins in shellfish muscle followed by UPLC-MS/MS analysis[j]. Talanta, 2013, 116: [26] Petrovic M, Hernando M D, Diaz-Cruz M S, et al. Liquid chromatography-tandem mass spectrometry for the analysis of pharmaceutical residues in environmental samples: A review[j]. Journal of Chromatography A, 2005, 1067(1):1-14. A solid phase extraction-ultrahigh performance liquid chromatography-tandem mass spectrometry method for the determination of lipophilic marine toxins in shellfish CHAI Ji-ye 1, WANG Lin 1, ZHAO Qiao-ling 2, HUANG Zhu-liang 2, SHI Xi-zhi 1*, SUN Ai-li 1, LI De-xiang 1 ( 1.School of Marine Sciences, Ningbo University, Ningbo , China; 2.Zhoushan Institute for Food and Drug Control, Zhoushan , China ) Abstract: A spin-mini solid phase extraction (SPE) based on the oxide graphene coupled with ultra performance liquid chromatography-electrospray ionization trap tandem mass spectrometry (UPLC-MS/MS) was developed to determine six lipophilic marine toxins (LPTs) in shellfish. Samples were firstly extracted with methanol: ethanol:isopropanol (7:2:1), purified with spin-mini solid phase extraction based on oxide grapheme. The analytes were analyzed by UPLC-MS/MS by an external standard method. The calibration curves of PTX2 and GYM showed good linearity in the range of μgkg -1. AZA1, AZA2, AZA3 showed good linearity in the range of μgkg -1. SPX1 showed good linearity in the range of μgkg -1, with linear correlation coefficient above The limits of detection (LODs) and limits of quantification (LOQs) were μgkg -1 and μgkg -1 respectively. The average recovery of 3 levels of 6 LPTs in shellfish samples was , and the relative standard deviation was The proposed method is efficient with low solvent consumption, high sensitivity, improved recovery and stability. The method can be used for the determination and verification of LPTs in shellfish. Key words: lipophilic marine toxins; ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS); oxide-graphene; solid phase extraction
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